Methods of lowering blink reflex for the treatment of dry eye disease

ABSTRACT

The present disclosure relates to, among other things, compositions and methods for treating an inflammatory condition including, but not limited to, ocular inflammation, dry eye disease, and ocular neuropathic pain. One aspect of the present disclosure relates to a composition comprising (a) chemerin or a fragment or analog thereof and (b) a lipid entity linked to the chemerin or fragment or analog thereof.

RELATED APPLICATIONS

This application is a National Stage Application, filed under 35 U.S.C.371, of International Application No. PCT/US2017/014605, filed on Jan.23, 2017, which claims priority to U.S. provisional patent applicationNo. 62/286,070, filed Jan. 22, 2016, the contents of each of theseapplications is incorporated by reference in its entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “OKYO-003/N01US-SequenceListing.txt,” which was created on Jul. 16, 2018 and is 3.59 KB in size,are hereby incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

There is a variety of inflammatory conditions including, but not limitedto, ocular inflammation, dry eye disease (DED), and ocular neuropathicpain.

Ocular inflammation can be caused by a microbial infection of the eye.Such infection may be fungal, viral, or bacterial. Ocular inflammationcan also be caused by trauma, burn, autoimmune disease, chemical injury,contact lens, or other external stimuli. Neuropathic pain is a majorhealth problem that occurs in as much as 7% of the general population.Up to 50% of patients do not respond to standard therapy.

DED is a multifactorial disease of the tears and the ocular surface withinflammation playing a part in its pathogenesis. Dry eye is a common andoften chronic problem, particularly in older adults. In 2000, itsprevalence in the US has been estimated around 17% in females and 12% inmales but it has been increased in recent years and estimated to be morethan 50%. People with dry eyes either do not produce enough tears ortheir tears are of a poor quality. Tears are produced by several glandsin and around the eyelids. Tear production tends to diminish with age,with various medical conditions or as a side effect of certainmedicines. Environmental conditions, such as wind and dry climates, canalso decrease tear volume due to increased tear evaporation. When thenormal amount of tear production decreases or tears evaporate tooquickly from the eyes, symptoms of dry eye can develop. As for thequality of tears, tears are made up of three layers: oil, water andmucus. Each component protects and nourishes the front surface of theeye. A smooth oil layer helps prevent evaporation of the water layer,while the mucin layer spreads the tears evenly over the surface of theeye. If the tears evaporate too quickly or do not spread evenly over thecornea due to deficiencies with any of the three tear layers, dry eyesymptoms can develop. The common form of dry eyes occurs when the waterlayer of tears is inadequate. This condition is also calledkeratoconjunctivitis sicca (KCS).

The present disclosure addresses the need of patients suffering fromvarious inflammatory conditions including, but not limited to, ocularinflammation, DED, and ocular neuropathic pain.

SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions and methods for treating orameliorating at least one symptom of an inflammatory condition.

In one aspect, the present disclosure relates to a compositioncomprising (a) chemerin or a fragment or analog thereof and (b) a lipidentity linked to the chemerin or fragment or analog thereof.

In some embodiments, the lipid entity is linked to the chemerin orfragment or analog thereof through a linker. The linker can comprisepolyethylene glycol, a peptide, or a combination thereof.

In some embodiments, the linker can be selected from the groupconsisting of:

In some embodiments, the chemerin fragment comprises at least 5 aminoacids, at least 10 amino acids, at least 20 amino acids, at least 30amino acids, at least 40 amino acids, at least 50 amino acids, at least60 amino acids, at least 70 amino acids, at least 80 amino acids, atleast 90 amino acids, at least 100 amino acids, at least 110 aminoacids, at least 120 amino acids, at least 130 amino acids, at least 140amino acids, or at least 150 amino acids, preferably of SEQ ID NO: 1.More preferably, the chemerin fragment retains CMKLR1 activationactivity.

In some embodiments, the chemerin fragment has an amino acid sequencefrom position 21 to 157 of SEQ ID NO: 1. SEQ ID NO: 1 corresponds to theamino acid sequence of chemerin.

In some embodiments, the chemerin fragment comprises YFPGQFAFS (SEQ IDNO: 2).

In some embodiments, the chemerin analog is resistant to proteolysis.For example, the chemerin analog comprises YFLPSQFA-Tic-S (SEQ ID NO:3), where the italicized Y, S, and A are D amino acids and Tic standsfor 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

In some embodiments, the lipid entity is selected from the groupconsisting of α-linolenic acid, γ-linolenic acid, stearidonic acid,eicosapentaenoic acid, docosahexaenoic acid, linoleic acid,dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid,palmitoleic acid, vaccenic acid, paullinic acid, oleic acid, elaidicacid, gondoic acid, erucic acid, nervonic acid, mead acid, myristicacid, palmitic acid, stearic acid,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), GM1ganglioside, GM2 ganglioside, GM3 ganglioside,1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a glycosphingolipid, asphingolipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), a ceramide,cholesterol, ergosterol, phytosterol, a hopanoid, and a steroid.

In some embodiments, the lipid entity is selected from the groupconsisting of α-linolenic acid, γ-linolenic acid, palmitic acid,vaccenic acid, oleic acid, and elaidic acid.

In some embodiments, the lipid entity can be linked at or near theN-terminus of the chemerin or fragment or analog thereof.

In some embodiments, the lipid entity can be linked at or near theC-terminus of the chemerin or fragment or analog thereof.

In another aspect, the present disclosure relates to a pharmaceuticalcomposition comprising the composition of the present disclosure and apharmaceutically acceptable carrier.

In yet another aspect, the present disclosure relates to a method oftreating an inflammatory condition in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of the pharmaceutical composition of the present disclosure or acomposition comprising chemerin or a fragment or analog thereof.

In some embodiments, the inflammatory condition is ocular inflammationor dry eye disease.

In some embodiments, the pharmaceutical composition is administeredtopically.

In some embodiments, the pharmaceutical composition is administered oncea day, twice a day, or thrice a day.

In some embodiments, the subject is a human.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein. While the disclosure has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the disclosure, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the luciferase assay. HEK293 cells weretransfected for 24 hours with cDNAs encoding: a) GPCR, b) a tetheredligand (where applicable), c) a luciferase reporter gene, and d)β-galactosidase (transfection control). Luciferase activity was measuredusing Steadylite reagent. Luciferase data were normalized toβ-galactosidase values.

FIG. 2 is a set of schematics showing membrane tethered ligands (MTLs)and synthetic membrane anchored ligands (SMALs). (Left) MTLs arerecombinantly expressed proteins that include a peptide ligand, anepitope tag, a linker sequence, and a transmembrane domain. (Right)SMALs are custom synthesized. Peptide ligands are conjugated to a PEGlinker and a lipid to create synthetic MTL analogs. Due to theirlipophilic properties SMALs anchor in the membrane when applied tocells.

FIG. 3 is a schematic depicting chemerin peptide processing. Chemerin(1-163) is cleaved on both the N and C-terminus during endogenousprocessing to generate a mature peptide (21-157) which is a knownagonist of the GPCR CMKLR1. A nine amino acid C-terminal fragment ofchemerin (149-157) (SEQ ID NO: 2) has also been reported as an activatorof CMKLR1. A series of chemerin peptides were incorporated into membranetethered ligand constructs.

FIGS. 4A-4B are graphs showing that membrane tethered chemerin activatesCMKLR1. Membrane tethered chemerin constructs encoding either maturechemerin (tChem21-157) or truncated chemerin (tChem149-157) bothactivate the (FIG. 4A) human CMKLR1 and (FIG. 4B) mouse CMKLR1.

FIGS. 5A-5D are graphs showing the luciferase activity of l-Chem149-157and s-Chem149-157. The lipidated chemerin peptide (1-Chem149-157) hasenhanced potency on both human and mouse CMKLR1 compared to its solublecounterpart (FIGS. 5A, 5C). L-Chem149-157 anchors in the membraneconferring wash resistance compared to its soluble counterpart(s-Chem149-157) (FIGS. 5B, 5D).

FIGS. 6A-6B are graphs showing that CMKLR1 agonists C16-stable Chem9 andResolvin E1 abrogate neuropathic pain in a chronic constriction injury(CCI) mouse model. Following intrathecal administration of compound,1-Stable Chem (C16-stable Chem9) blocked mechanical hypersensitivity formore than 24 hours after compound administration (FIG. 6A). In contrast,Resolvin E1 lost efficacy by 3 hours post injection (FIG. 6B).

FIG. 7 shows lipidated peptide equilibrium. In addition to peptideaffinity for its cognate receptor CMKLR1, in the context of systemicadministration, lipidated peptides may also bind to both the plasmamembrane and serum albumin. Changing the lipid tail therefore couldalter the equilibrium between these various bound states.

FIGS. 8A-8D are graphs showing that bovine serum albumin (BSA)differentially alters the pharmacological properties of lipidated stableChemerin 9 analogs. Wash resistance (an index of membrane adherence) ofpalmitic acid stable Chem9 is decreased in the presence of BSA (FIGS.8A-8B). With a linolenic acid (18C:3) stable Chem 9 analog, activity isfurther reduced (FIGS. 8C-8D) after washing in the presence of BSA.

FIGS. 9A-9D are graphs showing that β-Arrestin recruitment followingstimulation with lipidated stable chemerin 9 analogs. Potency andmaximum efficacy are altered by lipid tail substitution. Activity wasassessed using a Link-Light™ CMKLR1 stable cell line which measuresβ-Arrestin recruitment.

FIGS. 10A-10B are photographs for clinical slit lamp exam 10 days afterdesiccation (dry eye disease (DED) mice). Vehicle is used in FIG. 10A.oTTx-010 is used in FIG. 10B. oTTx-010 stands forpalmitate-PEG₈KGG-H₂N-Y*-F-L-P-S*-Q-F-A*-Tic-S-COOH (SEQ ID NO: 3), *denoting D amino acids and Tic stands for1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

FIG. 11 is a graph showing that corneal fluorescein staining scores werelower in DED mice treated with oTTx-010. NEI scoring system: 0-15.

FIG. 12 is a graph showing that blink reflex as a sign of ocularirritation was significantly lower in the oTTx-010 treated group ascompared to vehicle treated group of DED mice.

FIG. 13 is a set of flow cytometry graphs showing that percentage ofactivated immune cells (MHCII+) in the draining lymph node of oTTx-010treated mice decreased compared to vehicle controls.

FIG. 14 is a set of flow cytometry graphs showing that T regulatorycells increased in the draining lymph node of oTTx-010 treated micecompared to vehicle (showing more tolerance). Flow cytometry of draininglymph nodes gated on CD45+ CD3+ and CD4+ (T cells).

FIG. 15 is a set of flow cytometry graphs showing that cell infiltrationdecreases after topical application of oTTx-010 compared to vehicle.Flow cytometry of pulled corneas was performed on day 3 post cautery.Mice were treated topically three times a day with 10 μL of oTTx-010 orvehicle for 3 days. 10 μL of 210 nanomolar oTTx-010 in 1×PBSsolution=4.2 micrograms oTTx-010/dose.

DETAILED DESCRIPTION OF THE DISCLOSURE

CMKLR1 is a G protein-coupled receptor which has been shown to modulatenociception. This receptor is expressed in glia, dorsal root ganglionneurons, and immune cells. The endogenous ligand (agonist) for CMKLR1 ischemerin, a 163 amino acid protein. Chemerin, also known as retinoicacid receptor responder protein 2 (RARRES2), tazarotene-induced gene 2protein (TIG2), or RAR-responsive protein TIG2 is a protein that inhumans is encoded by the RARRES2 gene. The amino acid sequence ofchemerin (Homo sapiens) is shown below in SEQ ID NO: 1.

NCBI Reference Sequence: NP_002880.1

(SEQ ID NO: 1) MRRLLIPLAL WLGAVGVGVA ELTEAQRRGL QVALEEFHKHPPVQWAFQET SVESAVDTPF PAGIFVRLEF KLQQTSCRKRDWKKPECKVR PNGRKRKCLA CIKLGSEDKV LGRLVHCPIETQVLREAEEH QETQCLRVQR AGEDPHSFYF PGQFAFSKAL PRS.

Chemerin is inactive as pre-prochemerin (having SEQ ID NO: 1) and isactivated through cleavage of the C-terminus and N-terminus (FIG. 3) toform a chemerin fragment having an amino acid sequence from position 21to 157 of SEQ ID NO: 1, which can function as an agonist for CMKLR1.This chemerin fragment has the following amino acid sequence:

(SEQ ID NO: 4) ELTEAQRRGL QVALEEFHKH PPVQWAFQET SVESAVDTPFPAGIFVRLEF KLQQTSCRKR DWKKPECKVR PNGRKRKCLACIKLGSEDKV LGRLVHCPIE TQVLREAEEH QETQCLRVQR AGEDPHSFYF PGQFAFS.

In one aspect, the present disclosure provides a composition comprising(a) chemerin or a fragment or analog thereof and (b) a lipid entitylinked to the chemerin or fragment or analog thereof. Without wishing tobe bound by theory, the pharmacological properties of chemerin or afragment or analog thereof can be modulated by the choice of the lipidentity. In some embodiments, the composition of the present disclosurecan function as an agonist of CMKLR1.

The chemerin fragment is a fragment of the amino acid sequence of SEQ IDNO: 1. The chemerin fragment can retain some or all of the biologicalfunctions of the amino acid sequence of SEQ ID NO: 4, e.g., functioningas an agonist of CMKLR1. In some embodiments, the chemerin fragmentcomprises at least 5 amino acids, at least 10 amino acids, at least 20amino acids, at least 30 amino acids, at least 40 amino acids, at least50 amino acids, at least 60 amino acids, at least 70 amino acids, atleast 80 amino acids, at least 90 amino acids, at least 100 amino acids,at least 110 amino acids, at least 120 amino acids, at least 130 aminoacids, at least 140 amino acids, or at least 150 amino acids. In someembodiments, the chemerin fragment comprises about 5-150 amino acids,about 5-120 amino acids, about 5-100 amino acids, about 5-80 aminoacids, about 5-50 amino acids, or about 5-30 amino acids. In someembodiments, the chemerin fragment has an amino acid sequence fromposition 21 to 157 of SEQ ID NO: 1. In some embodiments, the chemerinfragment comprises YFPGQFAFS (SEQ ID NO: 2).

The chemerin analog can be an analog of either the full length orfragment of chemerin. The chemerin analog can retain some or all of thebiological functions of the amino acid sequence of SEQ ID NO: 4, e.g.,functioning as an agonist of CMKLR1. The chemerin analog can comprise atleast one amino acid modification, at least two amino acidmodifications, at least five amino acid modifications, or at least tenamino acid modifications. In some embodiments, the amino acidmodification is amino acid substitution. The chemerin analog can be moreresistant to proteolysis compared to the unmodified polypeptide. In someembodiments, the chemerin analog comprises YFLPSQFA-Tic-S (SEQ ID NO:3), wherein the italicized Y, S, and A are D amino acids and Tic standsfor 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. The chemerinanalog of SEQ ID NO: 3 is found to be resistant to proteolysis. SeeShimamura et al., “Identification of a stable chemerin analog withpotent activity toward ChemR23,” Peptides 30, 2009, 1529-1538, thecontents of which are incorporated by reference.

Any of a variety of lipid entities may be utilized in accordance withthe present disclosure. According to various embodiments, a lipid entitycan comprise an entity capable of insertion into a lipid bilayer (e.g.,a cell membrane). In some embodiments, a lipid entity is capable ofincorporating into a lipid raft in a lipid bilayer (e.g., a cellmembrane).

In some embodiments, the lipid entity can comprise a saturated orunsaturated fatty acid. The numbers in the lipid name are used todescribe the fatty acid chains on the lipid. The numbers are generallypresented in the format (number of carbons in fatty acid chain):(numberof double bonds in fatty acid chain), e.g., 16:0 would be 16 carbons inthe fatty acid chain with zero double bonds. The saturated orunsaturated fatty acid can include at least 4 carbons, at least 5carbons, at least 6 carbons, at least 7 carbons, at least 8 carbons, atleast 9 carbons, at least 10 carbons, or at least 15 carbons in thefatty acid chain. In some embodiments, the saturated or unsaturatedfatty acid can include about 4-24 carbons in the fatty acid chain. Thenumber of double bonds in the fatty acid chain can be in the range of0-10, e.g., 0-8, 0-6, 1-8, 1-6. For example, the lipid entity can beC22:0, C22:1, C22:2, C22:3, C22:4, C22:5, C22:6, C20:0, C20:1, C20:2,C20:3, C20:4, C20:5, C20:6, C18:0, C18:1, C18:2, C18:3, C18:4, C18:5,C18:6, C10:0, C10:1, C10:2, C10:3, C10:4, etc.

For example, the lipid entity can be selected from the group consistingof α-linolenic acid, γ-linolenic acid, stearidonic acid,eicosapentaenoic acid, docosahexaenoic acid, linoleic acid,dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid,palmitoleic acid, vaccenic acid, paullinic acid, oleic acid, elaidicacid, gondoic acid, erucic acid, nervonic acid, mead acid, myristicacid, palmitic acid, stearic acid,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), GM1ganglioside, GM2 ganglioside, GM3 ganglioside,1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a glycosphingolipid, asphingolipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), a ceramide,cholesterol, ergosterol, phytosterol, a hopanoid, a steroid,fluorinated-GM1, fluorinated-GM2, and fluorinated-GM3. In someembodiments, the lipid entity can be α-linolenic acid. In someembodiments, the lipid entity can be γ-linolenic acid. In someembodiments, the lipid entity can be palmitic acid. In some embodiments,the lipid entity can be vaccenic acid. In some embodiments, the lipidentity can be oleic acid. In some embodiments, the lipid entity can beelaidic acid.

The attachment of a lipid entity to a polypeptide is referred to aslipidation. In some embodiments, lipidation may compriseN-myristoylation. As used herein, “N-myristoylation” refers to theattachment of a myristate to an N-terminal glycine.

In some embodiments, lipidation may comprise palmitoylation. As usedherein “palmitoylation” refers to the creation of a thioester linkage oflong-chain fatty acids on one or more cysteine residues present in apeptide or protein.

In some embodiments, lipidation comprises GPI-anchor addition. As usedherein “GPI-anchor addition” refers to the linkage ofglycosyl-phosphatidylinositol (GPI) to the C-terminus of a protein.

In some embodiments, lipidation comprises prenylation. As used herein“prenylation” refers to the creation of a thioether linkage of anisoprenoid lipid (e.g., farnesyl (C-15) or geranylgeranyl (C-20)) to acysteine present in a peptide or protein. In some embodiments,lipidation comprises geranylation. In some embodiments, lipidationincludes geranylgeranylation. In some embodiments, lipidation comprisesthe association of a ligand entity with any compound that is soluble ina cellular membrane (e.g., 10:1 in equilibrium constant K_(assoc)≥10).

In some embodiments, lipidation may comprise one or more of thefollowing: attachment of diacylglycerol to the side chain of anN-terminal cysteine of a peptide or protein via the sulfur atom;attachment of O-octanoyl to a serine or threonine of a peptide orprotein; and attachment of S-archaeol to a cysteine of a peptide orprotein. In some embodiments, lipidation may occur, for example, at anylysine, glutamic acid, aspartic acid, serine, threonine, cysteine,and/or tyrosine. In some embodiments where a chemerin analog comprisesone or more ornithine, lipidation may occur at any ornithine.

In some embodiments, the lipid entity can be linked at or near theN-terminus of chemerin or fragment or analog thereof. In someembodiments, the lipid entity can be linked at or near the C-terminus ofchemerin or fragment or analog thereof.

In some embodiments, lipidation may include fluorination. Fluorinationcan include the addition of one or more C₆F₁₃ chains. Without wishing tobe bound by theory, it is thought that the presence of one or more C₆F₁₃chains may allow a lipid entity to segregate from hydrocarbon lipidmembrane components (see J. Am. Chem. Soc. 2007, 129, 9037-9043; J.Phsy. Chem. B, 2008, 112, 8250-8256; J. Am. Chem. Soc., 2009, 131,12091-12093).

In some embodiments, the presence of at least one alkene in thestructure of a lipid entity provides increased fluidity in a membrane(i.e., greater ability to move within the membrane) as compared tosimilar lipid entities lacking at least one alkene. In some embodiments,a lipid entity with greater fluidity is able to provide enhancedactivity towards targets (e.g., receptors, ion channels, or enzymes)with a low density in a membrane. Without wishing to be bound by theory,it is possible that lipid entities with increased ability to move withina membrane are able to encounter a low density target faster than alipid entity with less mobility within a membrane.

The composition of the present disclosure can optionally comprise alinker that links the lipid entity to chemerin or the fragment or analogthereof. For example, the linker can have a length of between about 2 Åand 175 Å, inclusive. In some embodiments, a linker is between 30 Å and150 Å, inclusive.

In some embodiments, the linker can comprise a peptide. In someembodiments, a peptide linker is between about 2 and 20 amino acidresidues in length. In some embodiments, a peptide linker is betweenabout 5 and 10 amino acid residues in length. According to variousembodiments, peptide linkers can be designed such that one or moreα-helices are formed between chemerin or a fragment or analog thereofand a lipid entity. In some embodiments, a peptide linker may comprise aplurality of α-helices. In some embodiments, the plurality of α-helicesis consecutive. In some embodiments, a plurality of α-helices is 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or more α-helices.

In some embodiments, a peptide linker can comprise repeating units, forexample a plurality of repeating glycine-asparagine (GN) units. In someembodiments, a peptide linker can comprise an epitope tag (e.g., a c-Myctag) or other marker to allow for identification and/or characterizationof provided agents and their fate in vitro and/or in vivo.

In some embodiments, the linker can comprise a non-peptide entity. Insome embodiments, non-peptide linkers may be a synthetic polymer.According to various embodiments, the synthetic polymer may be any of avariety of lengths. In some embodiments, a linker comprising a syntheticpolymer comprises a monomeric unit of the polymer. In some embodiments,a linker comprising a synthetic polymer comprises two or more monomericunits of a synthetic polymer (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 100 or more monomeric units).

In some embodiments, a linker can comprise at least one molecule ofpolyethylene glycol (PEG). Specific, non-limiting examples of suitablepolymeric linkers include linkers with one or more monomeric unitsaccording to one of the following formulas:

where n represents an integer greater than or equal to 1. In someembodiments, n is an integer between 2 and 50, 4 and 24, and/or 8 and24, inclusive.

In some embodiments, the linker can have any one of the followingstructures:

In some embodiments, a linker can comprise1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide (EDAC),Benzophenone-4-Isothiocyanate,Bis-((N-Iodoacetyl)Piperazinyl)Sulfonerhodamine, Succinimidyl2-(2-Pyridyldithio)Propionate (SPDP), 4-Azido-2,3,5,6-Tetrafluorobenzoicacid (ATFB), (N-((2-Pyridyldthio)ethyl)-4-Azidosalicylamide),Succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC),and/or N-(t-BOC)-aminooxyacetic acid. Those of skill in the art will beable to identify additional candidate linkers according to knownmethods.

In some embodiments, a linker can comprise both a peptide and anon-peptide entity.

In some embodiments, a linker is formed, at least in part, as a resultof a click reaction as further described below. In some embodiments, theclick reaction is an azide-alkyne Huisgen cycloaddition reaction.

Additional examples of lipid entities, linkers, and methods oflipidation can be found at US20160052982, the contents of which areincorporated herein by reference.

The composition of the present disclosure can be formulated into apharmaceutical composition, which can further comprise apharmaceutically acceptable carrier. Techniques for formulation of thedisclosed compositions can be found in Remington: the Science andPractice of Pharmacy, 19th edition, Mack Publishing Co., Easton, Pa.(1995). The pharmaceutical composition can be formulated for a varietyof administration routes.

In some embodiments, the pharmaceutical composition can be formulatedfor topical administration. Formulations suitable for topicaladministration include, but are not limited to, liquid and/or semiliquid preparations such as liniments, lotions, oil in water and/orwater in oil emulsions such as creams, ointments and/or pastes, and/orsolutions and/or suspensions. Topically-administrable formulations may,for example, comprise from about 1% to about 10% (wt/wt) activeingredient, although the concentration of active ingredient may be ashigh as the solubility limit of the active ingredient in the solvent.Formulations for topical administration may further comprise one or moreof the additional ingredients.

In some embodiments, the pharmaceutical composition can be formulatedfor oral administration. Oral formulations containing the pharmaceuticalcomposition described herein can be formulated into any conventionallyused oral forms, including: tablets, capsules, pills, troches, lozenges,pastilles, cachets, pellets, medicated chewing gum, granules, bulkpowders, effervescent or non-effervescent powders or granules,solutions, emulsions, suspensions, solutions, wafers, sprinkles,elixirs, syrups, buccal forms, and oral liquids. Capsules may containmixtures of the active compound(s) with inert fillers and/or diluentssuch as the pharmaceutically acceptable starches (e.g. corn, potato ortapioca starch), sugars, artificial sweetening agents, powderedcelluloses, such as crystalline and microcrystalline celluloses, flours,gelatins, gums, etc. Useful tablet formulations can be made byconventional compression, wet granulation or dry granulation methods andutilize pharmaceutically acceptable diluents, binding agents,lubricants, disintegrants, surface modifying agents (includingsurfactants), suspending or stabilizing agents, including, but notlimited to, magnesium stearate, stearic acid, talc, sodium laurylsulfate, microcrystalline cellulose, carboxymethylcellulose calcium,polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum,sodium citrate, complex silicates, calcium carbonate, glycine, dextrin,sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose,kaolin, mannitol, sodium chloride, talc, dry starches and powderedsugar. In some embodiments are surface modifying agents which includenonionic and anionic surface modifying agents. For example, surfacemodifying agents include, but are not limited to, poloxamer 188,benzalkonium chloride, calcium stearate, cetostearyl alcohol,cetomacrogol emulsifying wax, sorbitan esters, colloidal silicondioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate,and triethanolamine. Oral formulations herein may utilize standard delayor time release formulations to alter the absorption of the activecompound(s). The oral formulation may also consist of administering theactive ingredient in water or a fruit juice, containing appropriatesolubilizers or emulsifiers as needed.

Methods of Treatment

The compositions described herein can be used to treat a variety ofinflammatory conditions including, but not limited to, ocularinflammation, dry eye disease (DED), and ocular neuropathic pain.

In one aspect, the present disclosure provides a method of treating aninflammatory condition in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of thepharmaceutical composition of the present disclosure or a compositioncomprising chemerin or a fragment or analog thereof.

In some embodiments, the inflammatory condition is ocular inflammation.In some embodiments, the ocular inflammation is uveitis. Uveitis is awide range of inflammatory diseases of the eye, specifically the uvea.There are 3 basic layers of the eye—the sclera and cornea on theoutside, the retina on the inside, and the uvea in between. The uvea iscomprised mostly of blood vessels and connective tissue, includingpigmented cells. The different parts of the uvea are the iris in thefront, the ciliary body in the middle, and the choroid located behindthese, which lies around most of the eye. Sometimes uveitis can affectparts of the eye other than uvea, such as retina, vitreous, or opticnerve. Types of uveitis are based on what part of the eye is affected.For example, anterior uveitis is the inflammation in the front of theeye, called iritis or iridocyclitis; intermediate uveitis is theinflammation in the middle part of the eye, or pars planitis orvitritis; posterior uveitis is the inflammation of the back of the eye,such as choroiditis, retinal vasculitis, retinitis, neuroretinitis,retinochoroiditis, or chorioretinitis.

Symptoms of uveitis commonly include redness, blurry vision, pain, lightsensitivity, and floaters and flashes.

Ocular inflammation can be diagnosed through a review of illnesshistory, slit lamp examination, blood work, or any combination thereof.

Current therapies for treating ocular inflammation include locallyadministered anti-cytokine or anti-inflammatory agents. In someembodiments, the pharmaceutical composition of the present disclosure ora composition comprising chemerin or a fragment or analog thereof can beadministered in combination with an anti-cytokine or anti-inflammatoryagent for treating ocular inflammation.

Anti-cytokine or anti-inflammatory agents include, but are not limitedto, NF Kappa B inhibitors, for example corticosteroids, glucocorticoidssuch as flucinolonone; nonsteroidal anti-inflammatory drugs (NSAIDs)such as sulindac and tepoxalin; antioxidants such as dithiocarbamate;and other compounds such as sulfasalazine[2-hydroxy-5-[-4-[C2-pyridinylamino)sulfonyl]azo]benzoic acid],clonidine, and autologous blood-derived products such as Orthokine.

In some embodiments, the inflammatory condition is DED. DED is primarilycaused by the break-down of the pre-ocular tear film which results indehydration of the exposed outer surface. People with DED may experienceirritated, gritty, scratchy or burning eyes; a feeling of something intheir eyes; excess watering; and blurred vision. The definition andclassification of DED can be found at “The Definition and Classificationof Dry Eye Disease: Report of the Definition and ClassificationSubcommittee of the International Dry Eye Workshop (2007),” the OcularSurface 2007, Vol. 5, 75-92, the contents of which are incorporatedherein by reference.

DED can be diagnosed through a comprehensive eye examination. Testing,with emphasis on the evaluation of the quantity and quality of tearsproduced by the eyes, may include: (a) patient history to determine thepatient's symptoms and to note any general health problems, medicationsor environmental factors that may be contributing to the dry eyeproblem; (b) external examination of the eye, including lid structureand blink dynamics; (c) evaluation of the eyelids and cornea usingbright light and magnification; and (d) measurement of the quantity andquality of tears for any abnormalities. Special dyes may be put in theeyes to better observe tear flow and to highlight any changes to theouter surface of the eye caused by insufficient tears.

Without wishing to be bound by theory, there is a rationale that ocularinflammation as a result of pro-inflammatory cytokines and growthfactors plays a major role in the underlying causes of DED. As such,locally administered anti-cytokine or anti-inflammatory agents are oftenused in the treatment of DED. In some embodiments, the pharmaceuticalcomposition of the present disclosure or a composition comprisingchemerin or a fragment or analog thereof can be administered incombination with an anti-cytokine or anti-inflammatory agent fortreating DED.

In some embodiments, the inflammatory condition is ocular neuropathicpain. Ocular neuropathic pain can be caused by inflammation. Therefore,it can be treated by the pharmaceutical composition of the presentdisclosure, optionally in combination with an anti-cytokine oranti-inflammatory agent. Neuropathic pain has typical symptoms likedysesthesias (spontaneous or evoked burning pain, often with asuperimposed lancinating component), but the pain may also be deep andaching. Other sensations like hyperesthesia, hyperalgesia, allodynia(pain due to a normoxious stimulus), and hyperpathia (particularlyunpleasant, exaggerated pain response) may also occur.

Methods of diagnosing inflammation in the eye can be found in Teoh andDick, “Diagnostic techniques for inflammatory eye disease: past, presentand future: a review,” BMC Ophthalmology 2013, 13:41, the contents ofwhich are incorporated herein by reference.

With respect to combination therapies involving a first therapeuticagent (e.g., a pharmaceutical composition of the present disclosure or acomposition comprising chemerin or a fragment or analog thereof) and asecond therapeutic agent (e.g., an anti-cytokine or anti-inflammatoryagent), the first therapeutic agent can be administered concurrentlywith the second therapeutic agent; the first therapeutic agent can beadministered before the second therapeutic agent; or the firsttherapeutic agent can be administered after the second therapeuticagent. The administrations of the first and second therapeutic agentscan be separated by minutes or hours, e.g., about one hour, two hours,three hours, four hours, five hours, or six hours.

The therapeutically effective amount of a composition according to thisdisclosure can vary within wide limits and may be determined in a mannerknown in the art. For example, the composition can be dosed according tobody weight. Such dosage will be adjusted to the individual requirementsin each particular case including the specific compound(s) beingadministered, the route of administration, the condition being treated,as well as the patient being treated. In another embodiment, the drugcan be administered by fixed doses, e.g., dose not adjusted according tobody weight. In general, a daily dosage of from about 0.5 mg to about1000 mg should be appropriate, although the upper limit may be exceededwhen indicated. The dosage can be from about 5 mg to about 500 mg perday, e.g., about 5 mg to about 400 mg, about 5 mg to about 300 mg, about5 mg to about 200 mg. The daily dosage can be administered as a singledose or in divided doses, or for parenteral administration it may begiven as continuous infusion. The pharmaceutical composition or acomposition comprising chemerin or a fragment or analog thereof can beadministered once a day, or several times a day, e.g., twice a day, orthrice a day.

A therapeutically effective amount of a composition is that whichprovides an objectively identifiable improvement as noted by theclinician or other qualified observer.

In some embodiments, a therapeutically effective amount for treatingocular inflammation is an amount that reduces the extent of inflammationin the subject by at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or at least 95% compared to a placebo.

In some embodiments, a therapeutically effective amount for treating DEDis an amount that increases the production of tears in the subject by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, orat least 150% compared to a placebo.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. Thecompositions described herein can be administered orally, nasally,transdermally, pulmonary, inhalationally, buccally, sublingually,intraperintoneally, subcutaneously, intramuscularly, intravenously,rectally, intrapleurally, intrathecally, topically, or parenterally. Inone embodiment, the composition is administered topically. For example,the composition is administered in the form of eye drops. One skilled inthe art will recognize the advantages of certain routes ofadministration.

The dosage regimen utilizing the compositions described herein isselected in accordance with a variety of factors including species,ethnicity, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularcomposition employed. An ordinarily skilled physician or veterinariancan readily determine and prescribe the effective amount of the drugrequired to prevent, counter, or arrest the progress of the condition.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although other methods andmaterials similar, or equivalent, to those described herein can be usedin the practice of the present invention, the preferred materials andmethods are described herein. It is to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein and typically refer to a molecule comprising achain of two or more amino acids (e.g., most typically L-amino acids,but also including, e.g., D-amino acids, modified amino acids, aminoacid analogs, and amino acid mimetic). Peptides may be naturallyoccurring, synthetically produced, or recombinantly expressed. Peptidesmay also comprise additional groups modifying the amino acid chain, forexample, functional groups added via post-translational modification.Examples of post-translation modifications include, but are not limitedto, acetylation, alkylation (including methylation), biotinylation,glutamylation, glycylation, glycosylation, isoprenylation, lipoylation,phosphopantetheinylation, phosphorylation, selenation, and C-terminalamidation. The term peptide also includes peptides comprisingmodifications of the amino terminus and/or the carboxyl terminus.Modifications of the terminal amino group include, but are not limitedto, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal carboxy group include, butare not limited to, amide, lower alkyl amide, dialkyl amide, and loweralkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl).The term peptide also includes modifications, such as but not limited tothose described above, of amino acids falling between the amino andcarboxy termini. The term peptide can also include peptides modified toinclude one or more detectable labels.

The phrase “amino acid residue” as used herein refers to an amino acidthat is incorporated into a peptide by an amide bond or an amide bondmimetic.

The terminal amino acid at one end of the peptide chain typically has afree amino group (i.e., the amino terminus or N terminus). The terminalamino acid at the other end of the chain typically has a free carboxylgroup (i.e., the carboxy terminus or C terminus). Typically, the aminoacids making up a peptide are numbered in order, starting at the aminoterminus and increasing in the direction of the carboxy terminus of thepeptide.

As used herein, the term “analog” refers to a variant or mutantpolypeptide having one or more amino acid modifications compared to thewild type.

As used herein, an “amino acid modification” refers to a change in theamino acid sequence of a predetermined amino acid sequence. Exemplarymodifications include an amino acid substitution, insertion and/ordeletion. An “amino acid modification at” a specified position, e.g. ofchemerin or a fragment thereof, refers to the substitution or deletionof the specified residue, or the insertion of at least one amino acidresidue adjacent the specified residue. By insertion “adjacent” aspecified residue is meant insertion within one to two residues thereof.The insertion may be N-terminal or C-terminal to the specified residue.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence withanother different “replacement” amino acid residue. The replacementresidue or residues may be “naturally occurring amino acid residues”(i.e. encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); isoleucine (lie): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). Substitution with one or more non-naturally occurring amino acidresidues is also encompassed by the definition of an amino acidsubstitution herein. A “non-naturally occurring amino acid residue”refers to a residue, other than those naturally occurring amino acidresidues listed above, which is able to covalently bind adjacent aminoacid residues(s) in a polypeptide chain. Examples of non-naturallyoccurring amino acid residues include norleucine, ornithine, norvaline,homoserine and other amino acid residue analogues such as thosedescribed in Ellman et al. Meth. Enzym. 202:301-336 (1991). To generatesuch non-naturally occurring amino acid residues, the procedures ofNoren et al. Science 244:182 (1989) and Ellman et al., supra, can beused. Briefly, these procedures involve chemically activating asuppressor tRNA with a non-naturally occurring amino acid residuefollowed by in vitro transcription and translation of the RNA. In someembodiments, an L amino acid can also be substituted by a D amino acid.

An “amino acid insertion” refers to the incorporation of at least oneamino acid into a predetermined amino acid sequence. While the insertionwill usually consist of the insertion of one or two amino acid residues,the present application contemplates larger “peptide insertions”, e.g.insertion of about three to about five or even up to about ten aminoacid residues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above.

An “amino acid deletion” refers to the removal of at least one aminoacid residue from a predetermined amino acid sequence.

The term “pharmaceutical composition” refers to a mixture of a compounddisclosed herein with other chemical components, such as diluents orcarriers. The pharmaceutical composition facilitates administration ofthe compound to an organism. Pharmaceutical compositions can also beobtained by reacting compounds with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers, preservatives, and adjuvants.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or a symptom associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder or symptomassociated therewith be completely eliminated. The terms “treat,”“treating,” or “treatment,” do not include prevention.

The term “therapeutically effective amount” refers to the amount of acompound that, when administered, is sufficient to prevent developmentof, or alleviate to some extent, one or more of the symptoms of thedisorder, disease, or condition being treated. The term “therapeuticallyeffective amount” also refers to the amount of a compound that issufficient to elicit the biological or medical response of a cell,tissue, system, animal, or human that is being sought by a researcher,veterinarian, medical doctor, or clinician.

As used herein, a “subject” can be any mammal, e.g., a human, anon-human primate, mouse, rat, dog, cat, cow, horse, pig, sheep, goat,camel. In a preferred embodiment, the subject is a human.

As used herein, a “subject in need thereof” is a subject having aninflammatory condition.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a solvent” includes a combination of two or moresuch solvents, reference to “a peptide” includes one or more peptides,or mixtures of peptides, reference to “a drug” includes one or moredrugs, reference to “a device” includes one or more devices, and thelike. Unless specifically stated or obvious from context, as usedherein, the term “or” is understood to be inclusive and covers both “or”and “and”.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

Unless specifically stated or obvious from context, as used herein, theterm “about” when used in conjunction with numerical values and/orranges generally refers to those numerical values and/or ranges near toa recited numerical value and/or range. In some instances, the term“about” can mean within ±10% of the recited value. For example, in someinstances, “about 100 [units]” can mean within ±10% of 100 (e.g., from90 to 110).

EXAMPLES Example 1

Abbreviations: s-Chem 21-157 (soluble recombinant human chemerincorresponding to amino acids 21-157); s-Chem 149-157 (soluble C-terminal9 amino acids of human chemerin); 1-Chem 149-157 (lipidated C-terminal 9amino acids of human chemerin); s-Stable Chem (soluble stable chemerinpeptide; 1-Stable Chem (lipidated stable chemerin peptide).

One emerging treatment strategy to treat neuropathic pain is to targetmodulators of the neuroinflammatory process which contributes toneuropathic pain. We have previously identified a palmitoylated stableanalog (“C16-Stable Chem9”) derived from a 9 amino acid fragment ofchemerin. This analog anchors to the cell membrane resulting in highpotency and long-term activity. When assessed in vivo, palm sC9alleviated neuropathic pain in a mouse model of chronic constrictioninjury. Extending from these observations, we hypothesized that thepharmacological properties of our peptide may be further optimized bymodifying the lipid anchor. We have chemically synthesized a series ofChem 9 analogs with various lipid tails (e.g. cholesterol, oleic acid,linolenic acid). In vitro pharmacological characterization of eachpeptide was done using HEK293 cells transiently expressing therecombinant CMKLR1. We have found that varying the lipid tail of theligand significantly alters agonist potency of chemerin 9 analogs(within a 30-fold range). Furthermore, selected lipid tails bias bindingto either albumin (thus enhancing systemic delivery) or to the cellularmembrane (which favors locally restricted function). The lipidatedCMKLR1 ligands have promise as modulators of neuropathic pain. Inaddition, our findings illustrate an approach where serialaddition/substitution of lipid anchors can be applied to optimize thepharmacological as well as pharmacokinetic properties of many peptideligands.

CMKLR1 β-Arrestin recruitment was assessed following a 1 hr treatmentwith stable Chem 9 analogs (FIGS. 9A-9D).

Chemerin a 136 amino acid protein can be truncated to a 9 amino acidpeptide which is active as either a membrane tethered ligand (MTL) or alipidated peptide.

A stabilized form of lipidated chemerin 9 is long acting in a mousemodel of neuropathic pain (FIGS. 6A-6B).

Altering the lipid tail of stabilized chemerin 9 alters thepharmacological properties in vitro in part by modifying adherence tothe plasma membrane and/or serum albumin binding.

Lipidated chemerin 9 derivatives can be further characterized in a CCImodel of neuropathic pain. Lipidated chemerin 9 analogs can be assessedin other mouse models of inflammation, e.g. dust mite model of asthma.Pharmacokinetics profiles of lipidated Chemerin 9 analogs can becompared in vivo. Lipidated peptides can be developed for other targetsthat modulate inflammation.

TABLE 1 Summary of the potencies of lipidated stable chemerin 9 analogs.Ligand mediated CMKLR1 canonical Gαi signal transduction varies(potencies assessed +/− wash and +/− albumin). EC50 (no EC50 (BSA) EC50(BSA) Lipid tail wash0 EC50 (wash x3) (no wash) (wash x3) C16  2.7 nM18.6 nM  6.4 nM  520 nM C18:1  4.7 nM 26.4 nM 12.7 2862 nM C18:3 45.7 nM 350 nM 73.3 nM ND C10 17.8 nM ND 17.8 nM ND C4 79.3 nM ND   11 nM NDC16:1 12.0 nM 42.2 29.5 3143 nM C14:1  9.3 nM  100 nM 26.2 nM 1821 nMCholesterol 84.5 nm  445 nM  108 nM 13460 nM  none 35.2 nM ND 5.1 nM ND

TABLE 2 Potencies and efficacies of lipidated stable chemerin analogs.Max Efficacy (% 10 μM Lipid tail EC50 s-Stable chem9) C16  3.23 nM164.8% C18:1  17.4 nM 159.7% C18:3 180.6 nM 110.1% C10 23.12 nM 98.29%C4 223.13 nM   89.7% C16:1 80.84 nM 153.6% C14:1 51.85 nM 123.6%Cholesterol 131.2 nM 149.3% none 31.92 nM   100% C16-CKK4-NH₂ ND ND(nonspecific peptide)

Example 2

Stable lipidated chemerin was assessed in mouse models of DED andcorneal inflammation.

In the DED mouse model, scopolamine is injected 3 times/day to decreasetear production in mice. Fenestrated cages and 4 fans around the cageworking 24 hours/day are used to induce desiccating stress and dry eyes.oTTx-010 is administered to the mice to assess treatment efficacy.Vehicle is administered as control. Results show that topical oTTx-010can be a potential treatment for the DED to decrease clinical symptomsand signs and improve immune tolerance (FIGS. 11-14).

Thermal cautery is a mouse model of corneal inflammation (see ArchOphthalmol. 2003 Hamrah et al.). Results show that topical oTTx-010 canbe applied as a new treatment to decrease inflammation in the eye (FIG.15).

What is claimed is:
 1. A method of decreasing blink reflex in a humansubject having dry eye disease, the method comprising topicallyadministering to an eye of the human subject a therapeutically effectiveamount of a composition, thereby decreasing the blink reflex, whereinthe composition comprises: a chemerin fragment consisting of thesequence of Y-F-P-G-Q-F-A-F-S(SEQ ID NO.: 2) or a chemerin analogconsisting of the sequence of Y*-F-L-P-S*-Q-F-A*-Tic-S (SEQ ID NO.: 3),wherein * denotes D amino acids and Tic represents1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; a lipid entity linkedto the chemerin fragment or chemerin analog, wherein the lipid entity isselected from the group consisting of α-linolenic acid, γ-linolenicacid, palmitic acid, vaccenic acid, oleic acid, and elaidic acid; and alinker connecting the lipid entity to the chemerin fragment or chemerinanalog, wherein the linker comprises polyethylene glycol, KGG, or acombination thereof.
 2. The method of claim 1, wherein the compositionis administered once a day, twice a day, or thrice a day.
 3. The methodof claim 1, wherein the composition is formulated for topicaladministration as eye drops.
 4. The method of claim 1, wherein the lipidentity is linked at or near the N-terminus of the chemerin fragment orchemerin analog.
 5. The method of claim 4, wherein palmitate-PEG₈KGG islinked at or near the N-terminus of the chemerin analog.